The broad long term objective of this proposal is to determine the molecular basis of superoxide production in human neutrophils. The proposed investigation will focus on the structure of human neutrophil flavocytochrome b and how it changes upon activation of the superoxide generating system. The fundamental assumption made in this proposal is that this cytochrome is the central electron transferase of superoxide production and that alterations of its structure will regulate the flow of electrons across the plasma membrane. Elucidation of the fundamental structural mechanism of regulation of this electron flow will provide crucial information necessary to understand the molecular basis of an essential process in phagocyte-mediated microbicidal killing and tissue injury. Examination of the structure/function relationships existing in this protein may lead to the development of rationally designed drugs that could ameliorate neutrophil mediated tissue damage and enhance neutrophil-mediated microbicidal killing. More specifically, this proposal outlines strategies for making novel monoclonal and recombinant antibodies recognizing native flavocytochrome b epitopes, defining their three dimensional structure in the 1-5delta range, and their placement in the protein in the 10 -60 delta range. It describes a plan to covalently modify flavocytochrome b in order to mark different sites on its surface with fluorescent probes, identify possible phosphorylation sites, and identify the locations of the heme binding sites. To precisely identify these modifications, it proposes to use HPLC combined with MALDI-TOF mass spectrometry and peptide sequencing of proteolytic digests of the modified proteins. Using these new probes and covalent modification of the flavocytochrome with fluorescent probes, it will be possible to map the protein surface relative to its primary structure and its intrinsic hemes using fluorescence resonance energy transfer. The proposal also outlines a strategy to use multidimensional NMR techniques including Transferred NOESY, Tr ROESY, TOCSY, ROSEY methods to determine the antibody-bound structures of peptide mimetics of the cytochrome surface. Finally a gene fragment phage expression library of the gp91 phox and p22phox genes will be produced to reveal flavocytochrome b structural elements that can be displayed on phage for screening against binding partners or monoclonal antibodies. Successful completion of this work will permit the development of a structural model of the cytochrome that will be able to incorporate its known sequence, transmembrane topology, and high resolution three dimensional structure of discrete surface sites.